Francis Impens

The miR-17-92 microRNA cluster regulates multiple components of the TGF-β pathway in neuroblastoma.

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-β signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-β signaling cascade as well as direct inhibition of TGF-β-responsive genes.

Listeria monocytogenes impairs SUMOylation for efficient infection.

During infection, pathogenic bacteria manipulate the host cell in various ways to allow their own replication, propagation and escape from host immune responses. Post-translational modifications are unique mechanisms that allow cells to rapidly, locally and specifically modify activity or interactions of key proteins. Some of these modifications, including phosphorylation and ubiquitylation, can be induced by pathogens. However, the effects of pathogenic bacteria on SUMOylation, an essential post-translational modification in eukaryotic cells, remain largely unknown. Here we show that infection with Listeria monocytogenes leads to a decrease in the levels of cellular SUMO-conjugated proteins. This event is triggered by the bacterial virulence factor listeriolysin O (LLO), which induces a proteasome-independent degradation of Ubc9, an essential enzyme of the SUMOylation machinery, and a proteasome-dependent degradation of some SUMOylated proteins. The effect of LLO on Ubc9 is dependent on the pore-forming capacity of the toxin and is shared by other bacterial pore-forming toxins like perfringolysin O (PFO) and pneumolysin (PLY). Ubc9 degradation was also observed in vivo in infected mice. Furthermore, we show that SUMO overexpression impairs bacterial infection. Together, our results reveal that Listeria, and probably other pathogens, dampen the host response by decreasing the SUMOylation level of proteins critical for infection.

A Role for SIRT2-Dependent Histone H3K18 Deacetylation in Bacterial Infection

Introduction Posttranslational modification of histones is a well-documented mechanism by which the chromatin structure is modulated to regulate gene expression. Increasing evidence is revealing the strong impact of bacterial pathogens on host chromatin. However, our knowledge of the mechanisms underlying pathogen-induced chromatin changes and the impact of histone modifications and chromatin modifiers on infection is still in its infancy. Mechanism and consequence of SIRT2 activation by L. monocytogenes. Listeria induces SIRT2 relocalization from cytoplasm to chromatin, where SIRT2 deacetylates H3K18. The consequences of this cascade are control of host transcription, as illustrated by representative genes regulated by SIRT2, and control of infection, as assessed by staining cells for the secreted bacterial factor InlC (red), which is overexpressed in the cytosol, and host actin, which is polymerized into comet tails by bacteria (green). Error bars indicate SEM; **P < 0.001. Ac, acetyl; deAc, deacetylase. Methods We used the model bacterium Listeria monocytogenes and analyzed the mechanisms underlying a specific histone modification, deacetylation of histone H3 on lysine 18 (H3K18). Through immunoblotting, mass spectrometry, and chromatin immunoprecipitation, we studied how infection affected this modification, both in vitro and in vivo. We used a combination of chemical inhibitors, small interfering RNA (siRNA), and knockout mice to discover the key role of the host histone deacetylase sirtuin 2 (SIRT2) and determine its effect on infection. We performed microarray analysis to identify how infection and SIRT2 modulated host transcription. Results L. monocytogenes induces deacetylation of H3K18. This modification is mediated by the host deacetylase SIRT2. Upon infection, SIRT2 translocates from the cytosol to the chromatin of the host at the transcription start sites of a subset of genes that are repressed. We find that this process is dependent on activation, by the bacterial protein InlB, of the cell surface receptor Met and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Finally, infecting cells in which SIRT2 activity was blocked (by pharmacological agents, treatment with siRNA, or the use of SIRT2–/– mice) resulted in a significant impairment of bacterial infection, showing that activity of SIRT2 is necessary for infection, both in vitro and in vivo. Discussion Our study identifies a stimulus, infection by L. monocytogenes, that leads to nuclear localization of SIRT2, a deacetylase previously shown to be mainly cytoplasmic. In fact, only upon infection and SIRT2 translocation from the cytoplasm to the chromatin does this deacetylase have a role in transcriptional repression. This mechanism of host subversion could be common to other invasive pathogens that induce deacetylation of histones, and it defines a target for potential therapeutic treatment. Bacterial Subversion Tactics Intracellular bacterial pathogens such as Listeria monocytogenes can change host cell transcription programs to promote infection. Eskandarian et al. (1238858) found that during infection, the Listeria effector protein InlB promoted the movement of a host protein deacetylase, SIRT2, from its normal location in the cytosol to the nucleus. In the nucleus, SIRT2 helped to repress a number of host cell genes by deacetylating one of their associated histones. In mice, reduced levels of SIRT2 impaired bacterial infection. The bacterial pathogen Listeria monocytogenes exploits histone modifications to reprogram its host. Pathogens dramatically affect host cell transcription programs for their own profit during infection, but in most cases, the underlying mechanisms remain elusive. We found that during infection with the bacterium Listeria monocytogenes, the host deacetylase sirtuin 2 (SIRT2) translocates to the nucleus, in a manner dependent on the bacterial factor InlB. SIRT2 associates with the transcription start site of a subset of genes repressed during infection and deacetylates histone H3 on lysine 18 (H3K18). Infecting cells in which SIRT2 activity was blocked or using SIRT2−/− mice resulted in a significant impairment of bacterial infection. Thus, SIRT2-mediated H3K18 deacetylation plays a critical role during infection, which reveals an epigenetic mechanism imposed by a pathogenic bacterium to reprogram its host.

Selecting protein N-terminal peptides by combined fractional diagonal chromatography

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines—which can include stable isotope labeling—occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography—tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ∼5 d.

Stable isotopic labeling in proteomics.

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon‐13, nitrogen‐15, and oxygen‐18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications.

Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli

Significance Small ubiquitin-related modifier (SUMO) is a posttranslational modification essential for many functions in eukaryotic cells. A better understanding of the role of this ubiquitin-like modification, identification of proteins modified by SUMO, and knowledge of the exact sites of SUMO conjugation are critical but remain experimentally challenging. We have developed an innovative proteomic strategy allowing proteome-wide identification of SUMOylation sites and quantification of cell SUMOylation changes in response to diverse stimuli. Identification of yet unknown SUMO targets and characterization of SUMOylome alterations in response to environmental stresses, drugs, toxins, or bacterial and viral infections will help decipher previously unidentified roles of SUMOylation in cell physiology and disease. SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.

MS-driven protease substrate degradomics

Proteolytic processing has recently received increased attention in the field of signal propagation and cellular differentiation. Because of its irreversible nature, protein cleavage has been associated with committed steps in cell function. One aspect of protease biology that boomed the past few years is the detailed characterization of protease substrates by both shotgun as well as targeted MS‐driven proteomics techniques. The most promising techniques are discussed in this review and we further elaborate on the bioinformatics challenges that accompany mainly qualitative, MS‐driven protease substrate degradome studies.

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

A Quantitative Proteomics Design for Systematic Identification of Protease Cleavage Events

We present here a novel proteomics design for systematic identification of protease cleavage events by quantitative N-terminal proteomics, circumventing the need for time-consuming manual validation. We bypass the singleton detection problem of protease-generated neo-N-terminal peptides by introducing differential isotopic proteome labeling such that these substrate reporter peptides are readily distinguished from all other N-terminal peptides. Our approach was validated using the canonical human caspase-3 protease and further applied to mouse cathepsin D and E substrate processing in a mouse dendritic cell proteome, identifying the largest set of protein protease substrates ever reported and gaining novel insight into substrate specificity differences of these cathepsins.

Mechanistic insight into taxol-induced cell death

We analysed the involvement of proteases during taxol-mediated cell death of human A549 non-small-cell lung carcinoma cells using a proteomics approach that specifically targets protein N termini and further detects newly formed N termini that are the result of protein processing. Our analysis revealed 27 protease-mediated cleavages, which we divided in sites C-terminal to aspartic acid (Asp) and sites C-terminal to non-Asp residues, as the result of caspase and non-caspase protease activities, respectively. Remarkably, some of the former were insensitive to potent pancaspase inhibitors, and we therefore suggest that previous inhibitor-based studies that report on the caspase-independent nature of taxol-induced cell death should be judged with care. Furthermore, many of the sites C-terminal to non-Asp residues were also uniquely observed in a model of cytotoxic granule-mediated cell death and/or found by in vitro cataloging human μ-calpain substrates using a similar proteomics technique. This thus raises the hypothesis that killing tumor cells by chemotherapy or by immune cells holds similar non-Asp-specific proteolytic components with strong indications to calpain activity.